Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

• Main Authors: Giuseppa De Luca, Barbara Cardinali, Lucia Del Mastro, Sonia Lastraioli, Franca Carli, Manlio Ferrarini, George A. Calin, Anna Garuti, Carlotta Mazzitelli, Simona Zupo, Mariella Dono
• Format: Article
• Language: English
• Published: MDPI AG 2020-06-01
• Series: International Journal of Molecular Sciences
• Subjects: Circulating Tumor Cells; breast cancer; liquid biopsy; Next-Generation Sequencing; molecular tagging; Whole Genome Amplification-free
• Online Access: https://www.mdpi.com/1422-0067/21/12/4364
• ISSN: 1661-6596; 1422-0067

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in <i>TP53</i>, <i>KRAS</i>, and <i>PIK3CA</i> genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.